We found previously that C57BL/6J (B6) and C3H/HeJ (C3) mice are susceptible and resistant, respectively, to lung injury induced by exposure to hyperoxia (100% oxygen). Using BXH Recombinant Inbred (RI) strains and a B6C3F2 cohort, we completed genome-wide screens for chromosomal loci of susceptibility genes that control hyperoxia-induced pulmonary injury. The analyses identified significant and suggestive susceptibility quantitative trait loci (QTLs) on chromosomes 2 and 3, respectively. A candidate gene within the chromosome 2 QTL is Nfe2l2 (nuclear factor, erythroid derived 2, like 2 or Nrf2), which encodes a transcription factor NRF2 (NF-E2 related factor 2). NRF2 has been identified as an antioxidant response element (ARE)-mediated positive regulator of detoxifying enzyme genes for protecting cells against electrophile toxicity, oxidative stress, and carcinogenicity. To begin testing the hypothesis that Nrf2 is a candidate gene for differential susceptibility to oxygen toxicity in B6 and C3 mice, sequence analysis of Nrf2 from B6 and C3 mice was also done to identify the potential molecular basis. A potentially important variation was identified: B6 mice possess a T to C substitution at -336, which is predicted to add a Sp1 transcription factor-binding site in B6 mice compared to C3. The polymorphism segregated with SSLP genotypes (D2Mit248 and D2Mit94) at the peak of the chromosome 2 QTL, and the susceptibility phenotype in B6C3F2 mice. These data suggested that the polymorphism is potentially an important determinant of susceptibility to hyperoxic lung injury in this model. We further tested the hypothesis that NRF2 contributes to pulmonary protection against hyperoxic injury in mice by exposing mice with site-directed mutation (knockout) of Nrf2 (Nrf2-/-) and wt mice (Nrf2+/+) to hyperoxia. Pulmonary injury was significantly greater in Nrf2-/- mice compared to Nrf2+/+ mice after 48 and 72 hr hyperoxia exposure. Hyperoxia also markedly elevated expression of NRF2 mRNA and DNA-binding activity of NRF2 in lungs of Nrf2+/+ mice.[unreadable] [unreadable] We have also begun to investigate whether Nrf2 is an important determinant of responsiveness to fibrogenic agents. Our studies have found that Nrf2 can protect against the severity of lung fibrosis that develops in response to bleomycin, and may suggest an alternative therapeutic strategy for this devastating disease.[unreadable] [unreadable] In collaboration with Sekhar Reddy (Johns Hopkins University) we have also been investigating the cell signaling pathway oxidant-induced Nrf2 activation. Because phosphoinositide-3-kinase (PI3K)/Akt signaling promotes cell survival, the significance of this pathway in mediating reactive oxygen species (ROS)-dependent hyperoxia-induced Nrf2 activation was investigated in C10 murine pulmonary epithelial cells. Results of these investigations suggest that EGFR-PI3K signaling through Akt and ERK kinases regulates ROS-dependent, hyperoxia-induced Nrf2 activation in pulmonary epithelial cells. In another related investigation, we have used primary cells isolated from the lungs of Nrf2(+/+) and Nrf2(-/-) mice to better understand the physiologic significance of Nrf2-induced redox signaling. Our studies were focused on type II cells because these cells are constantly exposed to the oxidant environment and play key roles in host defense, injury, and repair processes. Using this system, we found that Nrf2 deficiency leads to defects in type II cell proliferation and greatly enhances cell sensitivity to oxidant-induced cell death. Glutathione (GSH) supplementation rescued these phenotypic defects associated with the Nrf2 deficiency. These results suggest that dysfunctional Nrf2-regulated GSH-induced signaling is associated with deregulation of type II cell proliferation, which contributes to abnormal injury and repair and leads to respiratory impairment.